Aqueous amine enables sustainable monosaccharide, monophenol, and pyridine base coproduction in lignocellulosic biorefineries

Thought-out utilization of entire lignocellulose is of great importance to achieving sustainable and cost-effective biorefineries. However, there is a trade-off between efficient carbohydrate utilization and lignin-to-chemical conversion yield. Here, we fractionate corn stover into a carbohydrate fraction with high enzymatic digestibility and reactive lignin with satisfactory catalytic depolymerization activity using a mild high-solid process with aqueous diethylamine (DEA). During the fractionation, in situ amination of lignin achieves extensive delignification, effective lignin stabilization, and dramatically reduced nonproductive adsorption of cellulase on the substrate. Furthermore, by designing a tandem fractionation-hydrogenolysis strategy, the dissolved lignin is depolymerized and aminated simultaneously to co-produce monophenolics and pyridine bases. The process represents the viable scheme of transforming real lignin into pyridine bases in high yield, resulting from the reactions between cleaved lignin side chains and amines. This work opens a promising approach to the efficient valorization of lignocellulose.

All the chemicals were used without any purification.Commercial Celluclast 1.5L (40 FPU/mL), Cellic CTec2 (84 FPU/mL), -glucosidase (β-G) from Aspergillus niger (power, ≥ 750 U/g), and sodium polystyrene sulphonates (SPS, standard for GPC) were supplied by Novozymes and Sigma-Aldrich, respectively.The filter paper activity of cellulase was determined according to Ghose et al. 1 Enzymatic hydrolysis lignin (EHL) purified from the corncob enzymatic residue was kindly provided by Shandong Longlive Bio-technology Co., Ltd.Corn stover (CS) was kindly provided by COFCO Bio-Energy (Zhaodong) Co., Ltd. and ground to pass through a 20-mesh screen.Corn cob residue (CCR) treated with dilute acid was purchased from Jinan Shengquan Group Share-Holding Co., Ltd.Deep eutectic solvent (DES) was prepared by mixing ChCl and glycerol (1:2, molar ratio), and the mixture was heated at 80 °C with stirring until a clear liquid form. 2 Fourier transform infrared spectrometer: The FTIR spectra of lignin were recorded on an FTIR spectrometer from Bruker (TENSOR 27) using the potassium bromide pellet technique.

X-ray diffraction (XRD):
All the lignocellulose samples were milled to pass through a 60-mesh screen prior to measurement.The XRD diffraction profile was obtained using a Bruker diffractometer (D8 advance) within the range of 10-40° at a scanning rate of 4°/min.Deconvolution, peak detection, and Gaussian fitting of the measured diffractogram were performed using the PeakFit software.The Bragg angles of 14.8°, 16.3°, 22.4°, and 34.5° were associated with cellulose .The peak at 20.5° corresponded to amorphous cellulose, with its half-peak width being twice that of the other peaks.The crystallinity index (CrI), indicative of the overall crystallinity in the biomass, was calculated by dividing the area of the crystalline peaks by the total area of the obtained XRD patterns.To minimize the influence of amorphous lignin and hemicelluloses, the CrI-to-cellulose content ratio was employed as an indicator for assessing the crystallinity of cellulose itself.
Elemental analysis: An Elemantar Vario EL cube elemental analyzer was used to determine the C, H, and N contents of lignin.
Contact angle: A contact angle meter (JC2000C1, Powereach, Shanghai, China) was used to measure the water contact angle dissimilarity on different lignin films.

Gel-permeation chromatograph (GPC):
The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of lignin were determined using a Waters e2695 system equipped with a 30 cm × 7.8 mm (L.× I.D.) TSKgel GMPWxl column (TOSOH, Tokyo).A variable 2489 UV/Vis detector at wavelengths of 280 nm and 254 nm was used for the detection.Utilizing a 0.1 M NaOH aqueous solution as the mobile phase allowed direct detection of the liquids from DEA and NaOH pretreatment before and after hydrogenolysis, without the need of additional post-treatments such as acetylation.The flow rate of eluent was maintained at 1.0 mL/min, and the column temperature was set to 30 °C.For establishing the calibration curve, sodium polystyrene sulphonate (SPS) standards (210-17000 Da) and guaiacylglycerolβ-guaiacyl ether were used as standards.A 3rd order fit type was employed to relate apparent molecular weight to retention time.
Quartz crystal microbalance with dissipation monitoring (QCM-D): Lignin films were prepared on goldcoated QCM sensors.Prior to film preparation, the QCM gold sensors were immersed in a solution of 25% ammonia, 30% hydrogen peroxide, and water (1:1:5, v/v/v) while being treated with an ultrasonicator.They were then rinsed with deionized water and dried under a flow of nitrogen gas.After the cleaning process, 2 wt% lignin solutions were prepared by fully dissolving lignin samples in ammonia solution.These prepared lignin solutions were then added onto the crystal sensors, and thin lignin films were coated onto the QCM sensors using a spin coater (WS-400Bz-6NPP-LITE, Mycro Technologies Corp., China).
The binding of cellulase (Celluclast 1.5L) to the lignin films was monitored in situ using a QCM-D (Q-Sense E1 instrument, Q-Sense AB, Sweden).The experiments were performed at 40 °C in a pH 4.8 acetate buffer (50 mM).Upon contact with the buffer solution, the film underwent swelling until equilibrium was reached.Subsequently, the enzyme solution (0.1 g/L) was continuously injected at a flow rate of 0.15 mL/min.Due to its robust stability and repeatability, the frequency shifts in the third overtone (Δf3) were commonly used to reflect the adsorption ability.The cellulase adsorption capacity on the lignin film is calculated as follows: Where Δm means the adsorption mass change, ngcm −2 ; C denotes the mass sensitivity constant with a value of ngcm -2 Hz -1 ; Δf3 denotes the change in the third overtone for frequency, Hz.

Scanning Electron Microscopy (SEM):
The morphology of CS was characterized using a scanning electron microscope (Hitachi SU8220, Japan) after sputtering with a layer of gold.

X-ray photoelectron spectroscopy (XPS):
The N content in lignin power was quantified using an X-ray photoelectron spectrometer (AXIS SUPRA, SHIMADZU).

High-Performance Liquid
Where IC9 denotes the integral value of the aromatic ring; IS2,6, IG2, and IH2,6 represent the integral value of S, G, and H units, respectively.According to the internal standard (IC9), the amount of linkages could be calculated by the following formula: Where X denotes the linkage amount per 100 C9 units; IX is the integral value of the α-position in A (-O-4), B (-5), and C (-) subunits.

Gas chromatography-mass spectrometric (GC-MS):
The prepared sample was analyzed using GC-MS with an Agilent 5975C-7890A series GC equipped with an HP-5ms capillary column (30 m  250 m, L. × I.D., 0.25 μm film thickness).The injection port was set at 300 °C, and helium was used as the carrier gas at a flow rate of 1 mL/min with a split ratio of 20:1.The following temperature-rising program was used: the GC oven temperature profile initially held at 50 °C for 2 min, then increased to 180 °C at a rate of 8 °C/min, followed by a ramp of 10 °C/min to 300 °C, where it was held for 5 min.

Gas chromatography with flame-ionization detection (GC-FID):
The prepared sample was analyzed using a GC (SHIMADZU GC-2018 series) equipped with an HP-5MS column and a flame ionization detector (FID).The injection temperature was set to 300 °C, and nitrogen was used as the carrier gas at a flow rate of 30 mL/min.The column temperature program was as follows: an initial isothermal temperature of 50 °C for 2 min, followed by a ramp at a rate of 8 °C/min up to 180 °C, then a ramp at a rate of 10 °C/min to 230 °C (maintained for 5 min), and finally a ramp at a rate of 10 °C/min to 280 °C, where it was held for 5 min.The yield of the identified phenolic monomer was determined based on an internal standard (n-decane) and the effective carbon number (ECN) method.The weight fraction of each compound in the sample was calculated using the following formula: TOF-MS was calculated using the published method for quantifying aromatic products. 3For pyridine derivatives, the GC-FID chromatogram only displayed peaks for 2-methyl-5-ethylpyridine and 3-ethyl-4methylpyridine due to limited resolution.As a result, quantification of pyridine derivatives was accomplished through the following approach: first, using GC-FID, the weight of 2-methyl-5-ethylpyridine and 3-ethyl-4-methylpyridine in the sample was determined based on an internal standard (n-decane) and the ECN method.Subsequently, the relative response factor (RRF) for the pyridine derivative in the GC  GC-TOF-MS contour chromatogram was calculated using the following equation: Where m1 and m2 are the mass of 2-methyl-5-ethylpyridine and 3-ethyl-4-methylpyridine in the sample, respectively; A1 and A2 are the peak area of 2-methyl-5-ethylpyridine and 3-ethyl-4-methylpyridine in the GC  GC-TOF-MS contour chromatogram; mIS denotes the mass of n-decane added, and AIS is the peak area of n-decane in the GC  GC-TOF-MS contour chromatogram.
In the end, the weight fraction of each identified pyridine derivative was calculated using the following formula: Where Ypyridine derivative is the weight fraction of pyridine derivative i; Ai is the peak area of pyridine derivative i in the GC  GC-TOF-MS contour chromatogram.
Techno-economic analysis (TEA) methodology: Techno-economic models include a conceptual level of process design to develop a detailed process flow diagram, rigorous materials and energy balance calculation, capital and project cost estimation, a discounted cash flow economic model, and the calculation of an MPSP. 4,5,6 Nobenzene oxidation method: Nitrobenzene oxidation of DEA-L and AL were conducted following previously established procedures.7 40 mg of dried lignin was dissolved in a mixture of 2 N NaOH (4 mL) and nitrobenzene (0.24 mL) in a Teflon-lined reactor and heated in an oven at 170 °C for 1 h.The reaction mixture was centrifuged and then extracted with ethyl acetate (30 mL × 3) to remove unreacted nitrobenzene.
The alkaline water layer was acidified to pH 2 with a 2 N HCl solution and extracted with ethyl acetate (30 mL × 3).The organic layer was washed with brine and dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation.The resultant products were dissolved in 2 mL of acetone with decane as an IS.Silylations of the sample solution were performed with BSTFA prior to GC-MS analyses.
Mass balance of DEA: The DEA consumed in residual and dissolved lignin was calculated based on the N content of DEA-treated corn stover (N: 0.23%) and DEA-L (N: 3%), respectively.The loss of 40% DEA was assessed by recording the weight changes in the overall material before and after pretreatments.To figure out DEA loss in hydrogenolysis, HPLC equipped with an RID was employed to analyze the DEA content variations in a 4% DEA solution under hydrogenolysis conditions.For the determination of DEA involved in the amination of hydrogenolysis products (including all aminated compounds), the dried hydrogenolysis products obtained through rotary evaporation and subsequent freeze-drying were subjected to an N elemental analysis (N: 5.2%).

Molecular electrostatic potential:
The computations were performed using Materials Studio 2017 software and its DMol3 module.All molecular geometries were optimized using the B3LYP level of theory with a maximum of 50 iterations.After analysis using DMol3, molecular electrostatic potential diagrams of DEA molecules in various solution environments were generated.
Molecular dynamics simulations: Materials Studio 2017 software and its Forcite module were used for the computations.The lignin model used consists of nine phenylpropanoid structural units and five types of representative interunit linkages, including -O-4, -O-4, -, -5, and 5-O-4. 8The optimized geometry of the lignin structure using B3LYP/6-31 G (d, p) level of theory in Gaussian 09 software is shown below.To study non-bonded interactions between lignin and DEA in a 40% v/v DEA solution, the system containing an amorphous cell with one lignin molecule, one hundred DEA molecules, and eight hundred water molecules was constructed.Periodic boundary conditions were applied, using the COMPASS force field and Ewald method to control electrostatic interactions.Geometry optimization for the constructed system was carried out with a maximum of 10,000 iterations.Then, the NVT ensemble with a period of 400 ps was used to further equilibrate the system.Finally, simulations ran for 2 ns under the NPT system at a temperature of 403 K.The last 500 ps of the simulation were extracted for analysis of the radial distribution function (RDF).

Biomass composition analysis
Determination of ash in biomass: About 0.6 g of biomass solid was placed into a muffle furnace.The muffle furnace was operated under programmed temperature conditions: ramp from room temperature to 105 °C and kept for 10 min, then ramp to 250 °C and hold for 30 min, and finally ramped to 575 °C and hold for 8 h.As it cooled, the ash remaining was collected and weighed.The ash content in raw CS was 9.0%.

Determination of structural carbohydrates and lignin in biomass:
Structural carbohydrate and lignin content in biomass were analyzed by two-step acid hydrolysis according to the standard laboratory analytical procedures (LAPs) of the National Renewable Energy Laboratory (NREL).In detail, 0.3 g of biomass solid was mixed with 3.0 mL 72wt% H2SO4 in the pressure bottle (100 mL), and the mixture was shaken at 200 rpm and 30 °C for 60 min.After that, the acid was diluted to a 4% concentration by adding 84.0 mL of deionized water, and the acid hydrolysis was performed in an autoclave at 121 °C for 60 min.
In order to correct sugar losses, 10 mL of sugar recovery standard (SRS) containing 1.2 g/L glucose and 0.6 g/L xylose was mixed with 348 L of 72 wt% H2SO4 and placed into the autoclave together with the test samples.After the reaction finished, 1 mL of the supernatant was taken for sugar and acid-soluble lignin concentration determination by an HPLC and an UV-Visible spectrophotometer (320 nm), respectively.
Acid insoluble residue was dried at 105 °C for 4 h at least, and the weight was recorded.The raw CS is composed of 39.7% cellulose, 22.5% hemicelluloses, 13.8% acid-insoluble lignin (AIL), and 1.8% acidsoluble lignin (ASL).Here, lignin content is the result after deducting ash content.The cellulose and AIL content of corn cob residue (CCR) are 70.8% and 25.9%, respectively.

Lignin isolation from biomass solids and pretreatment liquors
Lignin isolation from biomass solids: The isolation of cellulolytic enzyme lignin (CEL) from untreated CS and the residual lignin from DEA-treated corn stover (DEA-RL) was conducted according to the published procedure. 9Briefly, the lignocellulosic solids were ground in a ball mill at 400 rpm for 4 h in total and in the intervals of 30 min working and 10 min pause.Subsequently, the ball-milled solid was enzymatically hydrolyzed with a Cellic® CTec2 dosage of 0.3 mL/g solid and 10% w/v biomass loadings for 24 h at 50 °C, pH 4.8, and 150 rpm.The hydrolyzed residues were then mixed with dioxane/water (9:1, v/v) at 35 °C for 48 h to extract lignin.After removing the solvent from the organic phase with a rotary evaporator, the rude lignin was redissolved in acetic acid/water (9/1, v/v), and then re-precipitated in plenty of water.Finally, the precipitations were water-washed, and subjected to freeze-drying to obtain target lignin samples.
Lignin isolation from pretreatment liquors: DEA-treated lignin (DEA-L) from the corresponding pretreatment liquor was isolated using the following procedure: The liquor was acidified with hydrochloric acid (10%) to pH = 2 and then centrifuged to collect the precipitate.After being washed with deionized water, the precipitate was freeze-dried to give DEA-L.
Alkaline lignin (AL) from the NaOH pretreatment liquor was isolated with the same procedure above.In brief, 5.4 g of CS was treated with 4% w/w NaOH at a biomass loading of 10% w/w and 130 °C for 1 h.
After the reaction, the solid substrate was separated from the NaOH pretreatment liquor and then washed twice with 10 mL of water per gram of solid.The NaOH pretreatment liquor was combined with the washings.The mixture was then acidified to isolate AL.

Supplementary Note 2: -O-4 model compound studies
In a 50-mL reactor, 25 mg of guaiacylglycerol-β-guaiacyl ether (GE) was mixed with 4 mL of a 40% v/v DEA aqueous solution.The reaction was carried out in an oven at 130 °C for 1 h, followed by drying with a rotary evaporator.0.8 mL of methanol was added to dissolve the dried residue, and then 120 L of the solution was sampled for silylation.In detail, the sampled solution was mixed with 500 L of N-methyl-Ntrimethyl-silyl-trifluoroacetamide (MSTFA), 400 μL of pyridine, and 220 L of the internal standard solution (10 mg decane dissolved in 10 mL methanol) and kept at 40 °C for 80 min.After the reaction, the sample was analyzed using GC-MS.However, due to the complexity of products and the presence of dimers, the NIST library was unable to identify some products.Therefore, these compounds were assigned based on a detailed analysis of their mass fragmentation patterns, as shown below.

Supplementary Note 3: Reaction of model compounds bearing C=O with amines in hydrogenolysis
In order to prove the hypothesis that pyridine bases were produced from the reaction between the side chains of aldehyde ketones in lignin and amine.Four model compounds bearing C=O groups, 4hydroxyacetophenone (15 mg), 4-hydroxy-3-methoxyphenylpyruvic acid (15 mg), 4-hydroxyphenylacetic acid (15 mg), and 4-hydroxy-3-methoxycinnamaldehyde (15 mg) were chosen and mixed with 40 mg of 10% Pd/C and 60 mg of copper acetate monohydrate in 20 mL of 5% v/v DEA.The hydrogenolysis conditions were as follows: 10 bar H2, 250 °C, 60 min, and 400 rpm.After hydrogenolysis, the resultant mixture was acidified to pH 2 with 10% HCl.Subsequently, ethyl acetate (40 mL  3) was added to extract phenolic monomers into the organic phase and leave N-containing monomers in the aqueous phase.The organic and aqueous phases were dried with rotary evaporation.3 mL of acetone and 0.5 mL of n-decane (1.0 mg/mL in methanol, internal standard) were added to dissolve the dried products from the organic phase, while the dried products from the aqueous phase were dissolved in 2 mL of acetone and 0.4 mL of internal standard.Then, magnesium sulfate anhydrous was added for dehydration.Finally, each sample was injected for GC-MS analysis after passing through a 0.22 μm membrane filter.The GC-MS results of products from the organic and aqueous phases are summarized below.
Chromatography (HPLC): An HPLC (Agilent 1260 Infinity Ⅱ) equipped with an HPX-87H column was used to determine the monosaccharide concentration.The program setup was as follows: 5 mM H2SO4 as an HPLC mobile phase, a flow rate of 0.5 mL/min, a column oven temperature of 50 °C, and a detector temperature of 50 °C.HPLC-MS: Analysis was performed on an Agilent1290 / Bruker maXis impact.The program for HPLC is shown below.The gradient elution program in HPLC-MS.Time (min) Methanol (v%) 0.1% Formic acid (v%) heteronuclear-single-quantum-coherence spectra (2D HSQC NMR): Lignin solutions were prepared by dissolving 100 mg of lignin power in 600 L of DMSO-d6.The 2D HSQC spectra of the lignin were recorded on a Bruker AVANCE III HD 600 MHz spectrometer with a scanning time of 8 h.The semi-quantitative assessment of lignin inter-linkages was conducted based on a cluster of signals representative of all C9 units.
Wdecane in sample (mg): the weight of decane added to each analyzed sample; MWdecane (mgmmol −1 ): the molecular weight of decane (142 mgmmol -1 ); ndecane (mmol): the molar amount of decane added in the sample; nmonomer (mmol): the molar amount of phenolic monomer in the sample; Amonomer in sample: the peak area of monomer in the GC-FID chromatogram; Adecane in sample: the peak area of decane in the GC-FID chromatogram; ECNdecane: the effective carbon number (10) of decane; ECNmonomer: the effective carbon number of the phenolic monomer; Yphenolic monomer (wt%): the weight yield of phenolic monomer; MWmonomer (mgmmol -1 ): the molecular weight of monomer;Wlignin in pretreatment liquor (mg): the weight of lignin in 20 mL DEA and NaOH pretreatment liquor are 68 mg and 83 mg, respectively.20 mL is the volume of pretreatment liquor used in hydrogenolysis.Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC  GC-TOF-MS):The sample was analyzed using an Agilent 8890 GC coupled with an Agilent 7250A TOF.The GC  GC is equipped with a DB-5MS column (60 m × 0.25 mm × 0.25 μm) as the first dimension column connected to a DB-17MS (1.0 m × 0.25 mm × 0.15 μm) as the second dimension column through an SV (C7-C40) connection.The GC system operated under programmed temperature conditions: starting at 40 °C for 5 min, ramping up to 310 °C at a heating rate of 3 °C/min, and holding at 310 °C for 20 min.The injection port was set at 300 °C, and helium served as the carrier gas at a constant flow rate of 1.0 mL/min without splitting.The modulation period was 7 s.Data acquisition was performed at a rate of 50 spectra per second within a scanning range of 30 to 650 amu.The GC-TOF-MS interface (transfer line) temperature was maintained at 280 °C, while the ion source temperature was set at 230 °C.TOF-MS detection was carried out in HES mode (−70 eV).The yield of N-containing aromatic monomer identified by GC  GC-